Loss of function of the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- and HCO3- channel, causes cystic fibrosis (CF). CF lung and intestinal disease are the most consequential disease manifestations. The most common mutation, deletion of phenylalanine 508 (F508del), disrupts CFTR processing and reduces the rate of channel opening. Increased CFTR activity underlies water and electrolyte losses in cholera toxin-induced diarrhea. For both diseases there is a need for better treatments that normalize CFTR channel function. CFTR and other ABC proteins have both ATPase and adenylate kinase activity. The traditional paradigm of CFTR function has been that opening and closing (gating) of the channel is coupled to ATPase activity. It is not known whether adenylate kinase activity contributes to CFTR function in vivo, and whether this activity is a meaningful target to treat CFTR-related diseases. In preliminary studies we made two pertinent discoveries. 1) We identified a CFTR mutation (Q1291F) that abolished adenylate kinase activity but had no significant effect on ATPase-dependent gating. It reduced Cl- channel activity in primary human airway epithelia. 2) We found that the adenylate kinase inhibitor Ap5A (P1,P5-di(adenosine-5') pentaphosphate) - in striking contrast to wild-type CFTR - increased channel activity of F508del CFTR. The objective of this application is to build on these preliminary data to ascertain a contribution of adenylate kinase-dependent CFTR gating in vivo and to provide a proof-of-concept that a compound interacting with the adenylate kinase active center might be a clinically useful potentiator of F508del CFTR. The central hypothesis is that normal CFTR function in disease-relevant organs, airways and intestine, relies on its adenylate kinase activity and that - as a consequence of a structural defect - Ap5A potentiates F508del CFTR channel activity through interactions with residue Q1291. In aim 1 we will use primary airway epithelia and examine the effects of Q1291F CFTR on HCO3- secretion and airway surface liquid (ASL) pH, which both play a pivotal role in the development of CF lung disease. We will also investigate whether 1) expression of Q1291F CFTR rescues the intestinal phenotype of CFTR-/- mice and 2) the mutation reduces cholera toxin-induced intestinal fluid losses. In aim 2 we will investigate how Ap5A interacts with F508del CFTR to potentiate channel activity using biochemical and electrophysiological approaches. These studies are expected to lead to new treatment approaches for CF and CFTR-dependent diarrheas. The proposed research is innovative because it addresses an understudied mechanism of CFTR gating, adenylate kinase activity, and seeks to shift the current paradigm of how CFTR functions in vivo. Furthermore, it builds on the unanticipated discovery that Ap5A potentiates F508del CFTR channel activity. Furthermore, the proposed research is relevant to NIH's mission to seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability.